Background and Long-term Objective: Known etiological agents are thought to account for about 19% of the estimated 76 million cases of foodborne disease in the U.S. annually, and less than 3% of estimated cases are specifically diagnosed. Little is known about the clinical presentation, epidemiology, or risk factors associated with the majority of recognized, suspected, or unknown foodborne pathogens. The Minnesota Department of Health, in conjunction with the University of Minnesota Center for Post-Harvest Food Protection and Defense, will build upon its extensive existing infrastructure in this area to conduct intensive microbiological, epidemiological, and medical investigation of representative populations to increase our understanding of the etiology and risk factors associated with foodborne disease, and improve our ability to conduct surveillance and outbreak investigations. Research Strategies: The populations under study will include patients with diarrheal disease visiting family practice clinics and associated urgent care facilities located in both primarily urban and primarily rural settings, patients calling our statewide foodborne disease hotline, and age/telephone dialing area matched well controls. The project will involve intensive epidemiological, medical, and microbiologic investigation to determine the etiology and risk factors for disease, and the development of novel diagnostic test methodologies. The initial phase will consist of testing and test development for known or suspected pathogens, and the second phase will include pathogen discovery strategies. All specimens, including those from diarrheal disease outbreaks of unknown etiology in Minnesota or other FoodNet sites during the study period, will be analyzed with the test panel and prepared for pathogen discovery assays. Specific Aims: Project activities will include: (1) determining the prevalence and significance of known or suspected foodborne disease pathogens with previously developed culture-based and non-culture detection methods, (2) developing and implementing diagnostic assays for an additional suspect pathogens without established detection methods, (3) identifying risk factors through case and control interviews using standard interview forms, (4) developing novel multiplex amplification techniques needed as a prerequisite for the practical use of liquid or fixed microarray platforms, (5) developing a multianalyte systems such as microarrays and/or Luminex LabMAP for simultaneous identification of large numbers of diarrheal pathogen targets, and (6) investigating antimicrobial susceptibility profiles in detected bacterial pathogens normal gut flora. Pathogen discovery efforts will be made during phase 2 of this grant through collaboration with other institutions and alternate funding mechanisms. Approaches will include high throughput 16S sequencing, viral family PCR using consensus-degenerate hybrid oligonucleotide primers, DNase SISPA methodology, and electron microscopy. Microbiological and epidemiological methods developed as part of this project will be designed so that they can be readily transferred to public health and clinical laboratories.